Determination of oxidized α-1-proteinase inhibitor in serum or plasma

ABSTRACT

A new method of determining oxidized α-1-proteinase inhibitor in serum or plasma for use in studying the development of chronic obstructive lung disease is disclosed. Levels of oxidized α-1-proteinase inhibitor indicate the potential for emphysema development with higher levels showing a decrease in lung protection against elastolytic enzymes such as elastase. This method can be used for patients with a potential for chronic obstructive lung disease rather than having to use bronchial lavage methods for such patients. No other method is known to exist for determining oxidized α-1-proteinase inhibitor in serum or plasma.

The Government has rights in this invention pursuant to grant No.HL26148 awarded by the Department of Health and Human Services.

BACKGROUND AND SUMMARY OF THE INVENTION

This invention relates to a method of determining oxidizedα-1-proteinase inhibitor in serum or plasma. This method is a diagnostictechnique useful in studying the development of chronic obstructive lungdisease. More specifically this method is useful for patients having apotential for chronic obstructive lung disease rather than usingbronchial lavage methods. No other method is known to exist fordetermining oxidized α-1-proteinase inhibitor in serum or plasma.

Pulmonary emphysema appears to be a consequence of accelerated lungelastin degradation that results from either an elevated level ofelastase activity or a decreased level of elastase inhibitor(s) withinthe lung, or a combination of both factors. Neutrophil elastase isprobably the major elastolytic activity involved in development ofemphysema although elastase activity from other sources such asmacrophages and monocytes may have a role. The principal inhibitor ofneutrophil elastase is the plasma protein α-1-proteinase inhibitor(formerly referred to as α-1-antitrypsin). Severe genetic deficienciesof this protein commonly result in emphysema in the affectedindividuals. α-1-proteinase prevents the accumulation of leukocyteswhich contain proteases such as elastase within their cytoplasmicgranules.

Most smokers who develop emphysema do so despite normal levels ofα-1-proteinase inhibitor. An explanation for emphysema in smokers withnormal α-1-proteinase inhibitor concentrations has evolved from theobservation of the ease of oxidation of the reactive site methionylresidue of this protein leading to a two-thousand fold reduced rate ofassociation of the oxidized inhibitor with human leukocyte elastase.Thus, reduced levels of elastase inhibitory activity can result frompartial oxidation of the α-1-proteinase inhibitor in the lung throughinhalation of oxidants present in tobacco smoke, ozone, or industrialgases, as well as from oxidants released from cells in the lung, even iflevels of plasma α-1-proteinase inhibitor are normal or elevated.

Chronic obstructive lung disease has been associated withα-1-antitrypsin (now referred to as α-1-proteinase inhibitor) deficiencywherein extraordinarily low levels of elastase inhibition were found inthe serum of the survey patients, assaying α-1-antitrypsin activity by astandard assay known in the art (Turino, G. M., Senior, R. M., Bhagwin,D. C., Keller, S., Levi, M. M., and Mandl, I., Science, 165:709, 1969).Release of proteolytic enzymes in lungs and the onset of emphysema havebeen linked to cigarette smoking (Hutchinson, D. C. S., Brit. J. Dis.Chest. 67:171, 1973). The relationship of leukocytic proteases to lungtissue degradation and α-1-antitrypsin inhibitor (now referred to asα-1-proteinase inhibitor) deficiency has been described which showed acorrelation between serum leukocytic elastase inhibitory capacity andserum trypsin inhibitory capacity wherein both trypsin and elastase wereinhibited by α-1-antitrypsin (Lieberman, J., Arch. Environ. Health27:196, 1973). The proteolytic enzyme elastase has been reported tocause irreversible lung damage after laboratory animals receivedintratrachial injections of pancreatic porcine elastase (Kaplan, P. D.,Kuhn, C., and Pierce, J. A., J. Lab. Clin. Med. 82:349, 1973). TheEriksson (Laurell, C. B. and Eriksson, S., Scand. J. Clin. Lab. Invest.15:132-134, 1963) method of emphysema diagnosis has been analyzed and anattempt has been made to determine the probability of the occurrence ofphenotypes predisposed to emphysema when low α-1-antitrypsin (nowreferred to as α-1-proteinase inhibitor) values were found usinggelatinous film or immunochemical methods (Pilacik, B. and Kowalczyk,J., Med. Pr. 30(3):207, 1979). The oxidant effect of cigarette smokinghas been reported wherein several mechanisms have been suggested bywhich tobacco smoke damages the lungs (Kimbel, P. and Kueppers, F., Ann.Intern. Med. 92(4):564, 1980). The concentration of proteases andantiproteases of the lower respiratory tract in normal individuals havebeen compared with those who smoke or have deficiency of serumα-1-antitrypsin (now referred to as α-1-proteinase inhibitor) wherein amechanism of elastase production within the alveoli of the lung has beensuggested (Gadek, J. E., Hunninghake, G. W., Fells, G. A., Zimmerman, R.L., Keogh, B. A., and Crystal, R. G., Bull. europ. Physiopath. resp. 16(Suppl.):27, 1980). Cigarette smoke has been described as an oxidant ofα-1-proteinase inhibitor wherein as little as three puffs of cigarettesmoke caused a significant decrease in the elastase inhibitor capacityper milligram of α-1-proteinase inhibitor (Janoff, A., Carp, H., andLee, D. K., Bull. europ. Physiopath. 16 (Suppl.):321, 1980). A cycle hasbeen proposed wherein proteolytic enzymes not only degrade tissueabnormally but also increase the production of oxidants to turn offinhibitor control of proteinases resulting in predicted rapid changes inproteinase levels and proteinase inhibitor levels (Travis, J., Beatty,K., Wong, P. S., and Matheson, N. R., Bull. europ. Physiopath resp. 16(Suppl.):341, 1980). A gel plate assay for the detection of elastaseactivity and for the measurement of serum elastase inhibitory capacityhas been reported which provides an indirect means for estimatingα-1-antitrypsin (now referred to as α-1-proteinase inhibitor) function(Billingsly, G. D. and Cox, D. W., Am. Rev. resp. Disease. 121(1):161-164, 1980).

The present invention describes a convenient method for determining thepercent of normal versus oxidized α-1-proteinase inhibitor in humanserum or plasma based on a measurable difference between the inhibitoryactivities of normal and oxidized α-1-proteinase inhibitor against atrypsin like enzyme and elastase. It is an object of the presentinvention to provide a method of determining oxidized α-1-proteinaseinhibitor in serum or plasma.

It is an object of the present invention to provide a method fordetermining the quantity of normal versus oxidized α-1-proteinaseinhibitor against a trypsin like enzyme and elastase.

It is an object of the present invention to provide a useful diagnostictechnique in studying the development of chronic obstructive lungdisease.

It is a further object to provide a useful diagnostic technique instudying patients having a potential for chronic obstructive lungdisease rather than using bronchial lavage methods.

These and other objects, aspects, and advantages of this invention willbecome apparent from a consideration of the accompanying specificationand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows interactions among macrophages, neutrophils, proteinases,and oxidants in the inactivation of α-1-proteinase inhibitors and thedevelopment of tissue damage. Elastases, which are a major type ofproteinases from macrophages and neutrophils, are normally inactivatedby α-1-proteinase inhibitor. In the absence of oxidants, α-1- proteinaseinhibitor is actived and complexes with elastases wherein the elastasesare inactived. Elastases increase the production of oxidants. In thepresence of oxidants, α-1-proteinase inhibitor is oxidized. Oxidizedα-1-proteinase inhibitor is an inactive inhibitor. In the absence ofactive α-1-proteinase inhibitor, elastases degrade tissue abnormallycausing tissue damage.

FIG. 2 is provided to show that α-2-macroglobulin, another major serumproteinase inhibitor which is known to inactivate proteolytic activityof serine proteinases without affecting esterolytic activity, does notaffect the method of this invention for measuring oxidizedα-1-proteinase inhibitor. FIG. 2 shows the effect of α-2-macroglobulin(α-2M) on the relative trypsin like inhibitory capacity (TIC)/elastaseinhibitory capacity (EIC) of a α-1-proteinase inhibitor (α-1PI). Theordinate is the relative trypsin like inhibitory capacity(TIC)/elastaseinhibitory capacity (EIC) of different concentrations of oxidizedα-1-proteinase inhibitor (α-1PI) (1.3 mg/ml) in the presence ofα-2-macroglobulin (α-2M) (2 mg/ml). The abscissa is the relative trypsinlike inhibitory capacity (TIC)/elastase inhibitory capacity (EIC) ofpartially oxidized preparations of α-1-proteinase inhibitor α-1PI) (1m/gml) in the absence of α-2-macroglobulin (α-2M).

GENERAL DESCRIPTION OF THE INVENTION

Levels of oxidized α-1 proteinase inhibitor indicate the potential foremphysema development with higher levels showing a decrease in lungprotection against elastolytic enzymes such as elastase.

"Trypsin like enzyme" is a term used to describe any enzyme which isinhibitable by α-1-proteinase inhibitor. Trypsin like enzymes includebut are not limited to the following enzymes: trypsin, chymotrypsin,thrombin, plasmin, cathepsin C, and acrosin.

An unknown sample of serum or plasma contains α-1-proteinase inhibitoras a component of the serum or plasma. Similarly, a standard reducedsample of serum or plasma contain α-1-proteinase inhibitor as acomponent of the serum or plasma.

A standard solution of a trypsin like enzyme refers to a solutioncontaining a known amount of trypsin like enzyme. Similarly, a standardsolution of an elastase refers to a solution containing a known amountof elastase.

The method for this invention for determining percent of oxidizedα-1-proteinase inhibitor, X, present in an unknown sample of serum orplasma comprises:

(a) assaying a portion of the unknown sample of serum or plasma forenzymatic activity of a trypsin like enzyme;

(b) assaying a portion of the unknown sample of serum or plasma forenzymatic activity of an elastase;

(c) selecting known standards and determining their activity for boththe trypsin like enzyme and elastase;

(d) determining an oxidized ratio, K_(o), of the unknown sample of serumor plasma wherein ##EQU1##

(e) determining a reduced ratio, K_(R), of a standard reduced sample ofserum or plasma, wherein ##EQU2##

(f) substituting the reduced ratio and the oxidized ratio into anequation ##EQU3## and solving the equation for X, the percent ofoxidized α-1-proteinase inhibitor.

Since sensitivity of measuring enzymatic activity of trypsin likeenzymes has been found to be relatively affected by actual enzymeconcentrations used, standards are selected that approximate the sample.More specifically, trypsin like enzyme concentrations are selected whichapproximate the unknown sample of serum or plasma. Similarly, sincesensitivity of measuring enzymatic activity of elastases has been foundto be relatively affected by actual enzyme concentrations used,standards are selected that approximate the sample. More specifically,elastase enzyme concentrations are selected which approximate theunknown sample of serum or plasma.

DETAILED DESCRIPTION OF THE INVENTION

The following description more particularly sets forth a method fordetermining percent of oxidized α-1-proteinase inhibitor present in anunknown sample based on a measurable difference between the inhibitoryactivities of normal and oxidized α-1-proteinase inhibitor against atrypsin like enzyme and elastase. Normal α-1-proteinase inhibitorinhibited both porcine trypsin and porcine pancreatic elastase. Oxidizedα-1-proteinase inhibitor lost its inhibitory activity towards porcinepancreatic elastase while its net porcine trypsin inhibitory capacity(trypsin like inhibitory capacity) was retained, although the rate ofassociation with trypsin was markedly reduced. Thus, the reduced ratio,K_(R), of a standard reduced sample of serum or plasma (net porcinetrypsin inhibitory capacity to net porcine elastase inhibitory capacity)of fully reduced α-1-proteinase inhibitor was a constant wherein itsvalue depended solely upon the assays used and the units of measurementas shown in equation (1), below. ##EQU4## Trypsin like inhibitorycapacity refers to a capacity to be inhibited by α-1-proteinaseinhibitor.

As α-1-proteinase inhibitor was progressively oxidized this ratiochanged to a higher value K_(o), an oxidized ratio of an unknown sampleof serum or plasma in proportion to the fractional loss of elastaseinhibitory capacity represented as X, which also equals the fractionaloxidation of α-1-proteinase inhibitor as shown in equation (2a) below.##EQU5## For an unknown sample K_(o) is determined as shown in equation(2b) below. ##EQU6## The more oxidized the α-1-proteinase inhibitor thehigher the ratio of trypsin like inhibitory capacity/elastase inhibitorycapacity became until the ratio approached infinity for completelyoxidized α-1-proteinase inhibitor. Substituting K_(R) from equation 1into equation 2 gave equation (3) below. ##EQU7## If the ratio K_(R) wasknown from a standard reduced sample of serum or plasma, and the ratioK_(o) of an unknown sample of serum or plasma was measured, the fractionof oxidized α-1-proteinase inhibitor in the unknown can be calculatedfrom a rearrangement of equation (3), above resulting in equation (4)below. ##EQU8## The percent of oxidized α-1-proteinase inhibitor, X, canbe determined by multiplying by 100 as shown in equation (5), below, andsolving for X, the percent of oxidized α-1-proteinase inhibitor.##EQU9##

METHODS

Venous blood was collected from healthy young volunteers, according to aprotocol approved by the Human Studies Committee of the Jewish Hospitalof St. Louis, St. Louis, Mo. The blood was allowed to clot overnight at4° C. and the serum removed by centrifugation. Ten of the twenty samplescollected were from individuals who regularly smoked one to two packs ofcigarettes per day. All of the smokers had been smoking within severalhours of blood collection.

N-chlorosuccinimide was obtained from the Aldrich Chemical Co.Benzoyl-L-Arginine ethyl ester was a product of Worthington BiochemicalsCorp. and was usually stored frozen as a stock solution of 30 mg/ml in90% acetonitrile, 10% dimethyl sulfoxide.Succinyl-L-alanyl-alanyl-alanyl-paranitroanilide was purchased fromBACHEM, Inc. and was stored as a 0.2 M stock solution in dimethylsulfoxide.

Porcine trypsin and porcine pancreatic elastase (Sigma Chemical Corp.)were prepared as stock solutions of 5 mg/ml in 10⁻³ M HCl and 0.05 MTris, 0.05 M NaCl, pH 8.0, respectively. Both solutions were storedfrozen. Human α-1-proteinase inhibitor and human α-2-macroglobulin wereprepared using methods of Pannell, R., Johnson, D., and Travis, J.,Biochemistry 13:5439, 1974 and of Virca, G. D., Travis, J., Hall, P. K.,and Roberts, R. C., Anal. Biochem. 89:274, 1978 which are known in theart. Oxidized α-1-proteinase inhibitor was prepared by the addition ofeight molar equivalents of N-chlorosuccinimide to the native protein atpH 9.0, followed by exhaustive dialysis against 0.05 M Tris-HCl, 0.05 MNaCl, pH 8.0 (7). Reduction of the oxidized protein was performed by themethod of Jori, G., Galiazzo, G., Marzotto, A., and Scoffone, E., J.Biol. Chem. 243:4272, 1968 which is known in the art using a 5% solutionof 2-mercaptoethanol, incubated with the protein for four days at pH 8.0and 23° C. The half time for reduction was one day. The reducedα-1-proteinase inhibitor was exhaustively dialyzed against 0.05 MTris-HCl, 0.05 M NaCl, pH 8.0, in a stoppered flask through whichnitrogen was bubbled to remove oxygen and was referred to as an exampleof a standard reduced sample of serum or plasma.

EXAMPLE I Assay for Porcine Trypsin Activity

Five microliters of stock trypsin solution was mixed with 100 μl of 0.2M Tris-HCl, pH 8.0, in a 3.0 ml cuvette. The volume was adjusted to 3.0ml by addition of 0.05 M TrisHCl, pH 8.0 and 20 μl of stockN-benzoyl-L-arginine ethyl ester as a substrate was then added (standardsolution of trypsin like enzyme). Trypsin activity was measured as thechange in absorbance at 253 nanometers per minute (ΔA₂₅₃ /min) using themethod of Schwert, G. W., and Takenaka, Y., Biochim. Biophys. Acta.16:570, 1955 which is known in the art.

EXAMPLE II

Assay for Porcine Pancreatic Elastase Activity

Five microliters of stock elastase solution was mixed with 100 μl of 0.2M Tris-HC1, pH 8.0, in a 3.0 ml cuvette. The volume was adjusted to 3.0ml by addition of 0.05 M Tris-HCl, pH 8.0, and 20 μl of stockSuccinyl-L-alanyl-L-alanyl-L-alanyl-paranitroanilide as a substrate wasthen added (standard solution of elastase). Elastase activity wasmeasured as the change in absorbance at 400 nanometers per minute (ΔA₄₀₀/min) using the method of Bieth, J., Speiss, B., and Wermuth, C. G.,Biochem. Med. 11:353, 1974 which is known in the art.

EXAMPLE III

Assay for Inhibitory Activity of α-1-proteinase Inhibitor

Twenty microliters of α-1-proteinase inhibitor or serum were mixed with100 μl of 0.2 M Tris-HCl, pH 8.0, in a 3.0 ml cuvette. Five microlitersof stock trypsin or porcine pancreatic elastase were then added. Afterthe mixture had incubated ten minutes, the volume was brought to 3.0 mlwith 0.05 M Tris-HCl, pH 8.0, and 20 μl of either stock trypsinsubstrate or stock elastase substrate was added. Residual enzymeactivity was then measured as described above. Inhibitory activities fortrypsin like enzyme (trypsin like inhibitor capacity) and elastase(elastase inhibitor capacity) were calculated by measuring activitydifferences between a standard enzyme solution alone and the samesolution to which α-1-proteinase inhibitor had been added as shown inequations (6a) and (6b), below. ##EQU10## For an unknown sample of serumor plasma, trypsin inhibitor capacity and elastase inhibitor capacitywere determined as shown in equations (6a) and (6b), above, wherein theunknown sample of serum or plasma contained the α-1-proteinaseinhibitor. For a standard reduced sample of serum or plasma, prepared asdescribed in Methods, above, trypsin like inhibitor capacity andelastase inhibitor capacity were determined as shown in equations (6a)and (6b), above, wherein the standard reduced sample of serum or plasmacontained the α-1-proteinase inhibitor.

Inactivation of α-1-proteinase inhibitor by the oxidizing agentN-chlorosuccinimide increased the trypsin like inhibitorycapacity/elastase inhibitory capacity ratio as shown in Table 1, below,for purified, mercaptoethanol-reduced α-1-proteinase inhibitor from 1.27to 130. The effect showed the utility of the assay in detecting oxidizedα-1-proteinase inhibitor in tissue samples. Substitution of the values1.27 for K_(R) and 130 for K_(o) in equation (4), above, gives theresult that 99% of the α-1-proteinase inhibitor was oxidized byN-chlorosuccinimide. The percent approached 100% which indicated theratio approached infinity.

When the trypsin like inhibitory capacity/elastase inhibitory capacityratios were compared in the serum samples of smokers and non-smokerssignificant differences, (P=0.005), were noted, see Tables 1 and 2,below.

                  TABLE 1                                                         ______________________________________                                        Inhibitory Activity Ratios of Native α-1-proteinase inhibitor           and Human Serum from Non-Smokers and Smokers.                                              ##STR1##                                                                                 ##STR2##                                              ______________________________________                                        α-1-PI (native, reduced)                                                              1.27         0                                                  α-1-PI (native, oxidized)                                                             130          99                                                 Serum (10 non-smokers)                                                                      1.25         0                                                  Serum (10 smokers)                                                                          1.60         23                                                 ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Inhibitory Activity Ratios of                                                 Serum from Non-Smokers and Smokers                                             ##STR3##                                                                                          Non-Smokers                                                                              Smokers                                       ______________________________________                                                     1.25         1.66                                                             1.24         1.48                                                             1.22         1.50                                                             1.11         1.26                                                             1.30         1.71                                                             1.14         1.81                                                             1.32         1.74                                                             1.14         1.53                                                             1.27         1.54                                                             1.44         1.63                                                Average      1.25         1.60                                                Std. Deviation                                                                             0.11         0.17                                                ______________________________________                                    

In the samples from non-smokers the average trypsin like inhibitorycapacity/elastase inhibitory capacity ratio, 1.25, matched the ratio of1.17 found for purified, mercaptoethanol-reduced α-1-proteinaseinhibitor indicating that α-1-proteinase inhibitor of non-smokers is ina chemically reduced state. Smokers' serum, however, contained oxidizedα-1-proteinase inhibitor, reflected by an average trypsin inhibitorylike capacity/elastase inhibitory capacity ratio of 1.60. Substituting1.60 for K_(o) and 1.25 for K_(R) in equation (5), above, leads to theresult that 23% of the α-1-proteinase inhibitor in smokers was oxidized.One non-smoker had a high ratio and one smoker had a low ratio. Thespecific reasons why these two individuals are exceptional are not yetknown but likely reflect other genetic patterns which influence the rateat which α-1-proteinase inhibitor is oxidized.

Measurement of the total α-1-proteinase inhibitor byimmunoelectrophoresis indicated that the average values of the serumfrom smokers was about 143% of normal, relative to a standard pool withthe non-smokers at the normal value. The findings of elevatedα-1-proteinase inhibitor in smokers is in agreement with resultspublished elsewhere.

The present invention offers several advantages in that (a) a ratio isinherently more sensitive than measurements based on a singleobservation; (b) the results are not influenced by the concentration ofα-1-proteinase inhibitor, nor by partial denaturation of α-1-proteinaseinhibitor, nor by the purity of the α-1-proteinase inhibitor; (c) theassays refer directly to a standard reduced sample of serum or plasmacontaining the α-1-proteinase inhibitor which is easily prepared; (d)any trypsin like enzyme such as porcine trypsin or bovine α-chymotrypsinand any known assay therefor are used; (e) similarly, any elastase andany known assay therefor are used; (f) the results convert directly topercent oxidation of α-1-proteinase inhibitor, and the data given inTables 1 and 2, above, show that the assay is useful over a broad rangeand sensitive enough to identify the effects of smoking onα-1-proteinase inhibitor.

Although the loss of elastase inhibitory capacity without acorresponding loss of trypsin like inhibitory capacity is typical ofoxidation of α-1-proteinase inhibitor, non-oxidizing reagents can alsoeffect a disproportionate loss of porcine elastase inhibitory capacityof human α-1-proteinase inhibitor and thus could interfere with theinterpretation of results obtained with this assay. For example, therewas a 70% loss of elastase inhibitory capacity with only a 30% loss oftrypsin inhibitory capacity after a twenty four hour incubation of humanα-1-proteinase inhibitor at pH 8.0, 37° C., in a 15 mM solution of themethylating agent, methyl iodide. Similarly, chemical modification ofthe tyrosyl residues of α-1-proteinase inhibitor with tetranitromethaneor N-acetylimidazole have been reported to result in a selective loss ofelastase inhibitory capacity and iodination has the same effect. Usingthe methods of the present invention, 23% of the α-1-proteinaseinhibitor in serum of smokers appears to be in the oxidized form. Thisvalue is in agreement with a published report that smokers' serumelastase inhibitory capacity was 20% less than expected of theimmunologically determined titer of serum α-1-proteinase inhibitor. Thepresent invention provides a method which is useful in studying thedevelopment of chronic obstructive lung disease such as emphysema. Thismethod is useful in predicting the occurrence of chronic obstructivelung disease such as emphysema in persons having a potential for chronicobstructive lung disease such as smokers.

The oxidation of α-1-proteinase inhibitor totally eliminates itsinhibitory activity for porcine elastase, while only significantlyaffecting its behavior toward trypsin like enzymes.

Porcine enzymes were used because human enzymes are not available;however, human enzymes, if available commercially, could be substitutedfor porcine enzymes with the same or better results.

EXAMPLE IV Effect of α-2-Macroglobulin

Since α-2-macroglobulin can inactivate the proteolytic activity ofserine proteinases without affecting esterolytic activity, the possibleinterfering effect of this inhibitor in the assays described above wasinvestigated. Samples of α-1-proteinase inhibitor at several differentdegrees of oxidation were made by mixing different proportions ofcompletely oxidized α-1-proteinase inhibitor with reduced α-1-proteinaseinhibitor. Each solution of partially oxidized inhibitor was diluted to1.3 mg/ml with buffer alone and to 1.3 mg/ml in the presence of 2 mg/mlof α-2-macroglobulin. At a concentration of 2 mg/ml theα-2-macroglobulin inhibited an equal volume of 1.0×10⁻⁶ M active porcinetrypsin. The concentrations of α-1-proteinase inhibitor andα-2-macroglobulin were chosen to approximate their concentrations inwhole plasma. Both α-1-proteinase inhibitor alone, as well asα-1-proteinase inhibitor mixed with 2 mg/ml of α-2-macroglobulin, wereassayed for trypsin like inhibitory capacity and elastase inhibitorycapacity. The trypsin like inhibitory capacity/elastase inhibitorycapacity ratios were then compared to determine if they were the samewith and without the added α-2-macroglobulin.

α-2-macroglobulin had no effect on the procedures described formeasuring oxidized -1-proteinase inhibitor levels. As shown in FIG. 2,there was no change in the trypsin like inhibitory capacity/elastaseinhibitory capacity ratio irrespective of the presence ofα-2-macroglobulin. Such a result can only be interpreted as meaning thatα-2-macroglobulin does not interfere in the assays. The data in thisfigure also depict the trypsin like inhibitory/elastase inhibitorycapacity ratios for varying percentages of oxidized α-1-proteinaseinhibitor.

EXAMPLE V Other Proteins in Plasma

In order to determine whether other proteins in plasma might interferewith the trypsin inhibitory capacity and elastase inhibitory capacityassays, experiments were performed in which samples of α-1-proteinaseinhibitor at several different degrees of oxidation were incubated withserum containing low levels of α-1-proteinase inhibitor in theconcentration of this inhibitor being less than 10% of normal. Assaysfor trypsin like inhibitory capacity and elastase inhibitory capacitywere then performed to determine if the addition of the partiallyoxidized inhibitor to this serum had any effect on the trypsin likeinhibitory capacity and elastase inhibitory capacity ratio.

Similarly, the effect of other plasma proteins on the trypsin likeinhibitory capacity and elastase inhibitory capacity assays was found tobe essentially negative. Again, the trypsin like inhibitorycapacity/elastase inhibitory capacity ratios did not change for variouspartially oxidized α-1-proteinase inhibitor samples when added toα-1-proteinase inhibitor deficient plasma and a pattern similar to thatshown in FIG. 2 was obtained.

The foregoing illustrates specific emodiments within the scope of thisinvention and is not to be construed as limiting said scope. It is to beunderstood that variations and modifications thereof may be made bythose skilled in the art without departing from the scope of theinvention.

What is claimed is:
 1. A method for determining percent of oxidizedα-1-proteinase inhibitor present in an unknown sample of serum or plasmacomprising:(a) assaying a first portion of the unknown sample of serumor plasma for enzymatic activity of an enzyme inhibitable byα-1-proteinase inhibitor; (b) assaying a second portion of the unknownsample of serum or plasma for enzymatic activity of an elastase; (c)selecting known standards and determining activity for both the enzymeinhibitable by α-1-proteinase inhibitor and the elastase in saidstandards; (d) determining an oxidized ratio, K_(o), of the unknownsample of serum or plasma wherein ##EQU11## wherein T_(k) =Enzymaticactivity of the enzyme inhibitable by α-1-proteinase inhibitor in thestandard solution of said enzyme;wherein T_(u) =Enzymatic activity ofthe enzyme inhibitable by α-1-proteinase inhibitor of an unknownsolution comprising the standard solution of said enzyme and the unknownsample of serum or plasma; wherein E_(k) =Enzymatic activity of theelastase in the standard solution of the elastase; and wherein E_(u)=Enzymatic activity of the elastase of an unknown solution comprisingthe standard solution of the elastase and the unknown sample of serum orplasma; (e) determining a reduced ratio, K_(R), of a standard reducedsample of serum or plasma, wherein ##EQU12## wherein T_(k) =Enzymaticactivity of the enzyme inhibitable by α-1-proteinase inhibitor in thestandard solution of said enzyme;wherein T_(r) =Enzymatic activity ofthe enzyme inhibitable by α-1-proteinase inhibitor in a known solutioncomprising the standard solution of said enzyme and the standard reducedsample of serum or plasma; wherein E_(k) =Enzymatic activity of theelastase in the standard solution of the elastase; and wherein E_(r)=Enzymatic activity of the elastase in a known solution comprising thestandard solution of the elastase and the standard reduced sample ofserum or plasma; (f) substituting the reduced ratio and the oxidizedratio into an equation ##EQU13## and solving the equation for X, wherebyX is the percent of oxidized α-1-proteinase inhibitor.
 2. A methodaccording to claim 1 wherein concentrations of the enzyme inhibitable byα-1-proteinase inhibitor are selected which approximate theconcentration of said enzyme in the unknown sample of serum of plasma.3. A method according to claim 2 wherein elastase concentrations areselected which approximate the concentration of elastase in the unknownsample of serum or plasma.
 4. A method according to claim 3 wherein theenzyme inhibitable by α-1-proteinase inhibitor is trypsin.
 5. A methodaccording to claim 1, wherein the enzymatic activity of the enzymeinhibitable by α-1-proteinase inhibitor is measured as the change inabsorbance at 253 nanometers per minute and wherein enzymatic activityof the elastase is measured as the change in absorbance at 400nanometers per minute.
 6. A method according to claim 5 wherein thestandard solution of the elastase is prepared with porcine pancreaticelastase.
 7. A method according to claim 6 wherein the standard solutionof the enzyme inhibitable by α-1-proteinase inhibitor is prepared withporcine trypsin.
 8. A method according to claim 6 wherein the standardsolution of the enzyme inhibitable by α-1-proteinase inhibitor isprepared with bovine α-chymotrypsin.
 9. A method according to claim 7wherein the standard reduced sample of serum or plasma is reduced bymercaptoethanol.
 10. A method according to claim 9 wherein the enzymaticactivity of the enzyme inhibitable by α-1-proteinase inhibitor ismeasured using Succinyl-L-alanyl-L-alanyl-L-alanyl-paranitroanilide as asubstrate.
 11. A method according to claim 10 wherein the enzymaticactivity of the enzyme inhibitable by α-1-proteinase inhibitor ismeasured using N-benzoyl-L-arginine ethyl ester as a substrate.
 12. Amethod according to claim 11 wherein the standard solution of theelastase comprises the elastase in 0.05 M Tris, 0.05 M NaCl, pH 8.0, ata concentration of 5 mg/ml.
 13. A method according to claim 11 whereinthe standard solution of the enzyme inhibitable by α-1-proteinaseinhibitor comprises said enzyme in 10⁻³ M HCl at a concentration of 5mg/ml.